Studying circadian (~24hr) rhythms offers a tremendous opportunity to understand a fundamental brain function at molecular, cellular and circuit levels as well as opportunities for novel therapies. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and is mutated in a familial sleep disorder. Clock genes function within pacemaker neurons in the brain to drive behavioral rhythms. Although we have a relatively good understanding of these molecular clocks, it is unknown how molecular clocks regulate rhythmic neuronal activity and how rhythmic outputs feed back to the molecular clock. To identify potential outputs of pacemaker neurons, we conducted gene expression profiling studies from a group of homogeneous master pacemaker neurons, the Drosophila LNvs. One gene rhythmically expressed in LNvs encodes an Inward rectifier K+ channel (Ir). In Aim 1, we test the idea that Ir expression is directly regulated by the core clock transcription factors to regulate LNv excitability. Altered Ir expression in LNvs changes the period length of circadian rhythms. Thus the electrical activity of LNvs feeds back to regulate their molecular clocks. To test if the mechanism for this is transcriptional, we measured gene expression profiles in LNvs that were either constitutively hyperpolarized or hyperexcited. We found dramatic re-organization of circadian gene expression such that hyperpolarizing LNvs at dawn when they are normally most excitable makes their circadian gene expression resemble dusk, when they are normally inactive. Conversely, hyperexciting LNvs at dusk makes their circadian gene expression resemble dawn. Thus LNv neuronal activity can impose time-of-day to circadian gene expression and this suggests a major revision to our understanding of circadian molecular clocks. In Aim 2, we propose to identify the transcription factor(s) and signaling pathway(s) that connect neuronal activity to circadian rhythms in gene expression. One group of genes mis-regulated by altering LNv excitability encode translation initiation regulators. This was striking because three protein translation regulators are highly expressed in LNvs and mutants in these factors alter circadian rhythms and/or clock resetting by light. Thus translation initiation is a previously unappreciated regulatory step in the molecular clock. In Aim 3, we propose to test if these translation regulators affect translation of specific clock mRNAs and/or global translation in LNvs. We will also test if these regulators respond to signals such as light and neuronal activity to acutely regulate translation in LNvs. We believe that the endogenous neural and molecular rhythms of LNvs, combined with techniques we developed for whole-genome profiling of LNvs from an intact nervous system, make LNvs a unique model to study the dynamic bi-directional relationships between gene expression and neuronal excitability and plasticity. PUBLIC HEALTH RELEVANCE: Disrupted circadian (~24hr) rhythms are associated with a wide range of human symptoms from sleep and mood disorders to increased incidences of cancer and obesity. These rhythms are controlled by intracellular clocks whose understanding at the molecular level may open up novel therapies. Here we seek to understand the molecules that regulate molecular clock speed via electrical activity and protein translation.